Fernández-Romero, José A. BS*; Thorn, Mitchell MS*; Turville, Stuart G. PhD*; Titchen, Kanani BS*; Sudol, Kristin BS*; Li, Jifan PhD*; Miller, Todd BS*; Robbiani, Melissa PhD*; Maguire, Robin A. BS*; Buckheit, Robert W. Jr PhD†; Hartman, Tracy L. MS†; Phillips, David M. PhD*
OUR LABORATORY HAS PREVIOUSLY DEVELOPED the topical microbicide, Carraguard, which contains the sulfated polysaccharide carrageenan. The inherent nature of carrageenan is such that it gives Carraguard the rheologic properties desirable for a vaginal microbicide1 and is itself an active antiviral and antibiotic ingredient. In vitro, carrageenan has been shown to block HIV-1.2 In animal systems, Carraguard has been shown to prevent: 1) rhesus macaques from vaginal infection by SHIV (R. Black, personal communication, and Stuart G. Turville, Todd Miller, and Melissa Robbiani, unpublished observations); 2) cell trafficking across the vaginal epithelium3; 3) vaginal infection by herpes simplex virus type 24; 4) vaginal colonization by Neisseria gonorrhoeae4; and 5) human papillomavirus from transforming human xenographs (M.K. Howett, personal communication). In addition, carrageenan has a strong safety profile5–7 and is remarkably stable.6,8,9 In 5 phase 1 and 2 expanded safety studies in humans, Carraguard was safe and acceptable when applied vaginally.10
In addition to carrageenan, several other sulfated polymers have been shown to be active in blocking HIV infection of target cells by T-tropic viruses in vitro.11–13 Given that these sulfated polymers are negatively charged molecules, it is believed that their mechanism of action is to bind to the positively charged region of the viral envelope.14–16 Because the envelope of CCR5 viruses (M-tropic) is less positively charged than CXCR4 viruses (T-tropic),17,18 there is controversy over whether sulfated polymers will be effective in blocking CCR5 viruses. Ability to block CCR5 viruses is critical for a microbicide, because even if both phenotypes are transmitted together, it is the CCR5 viruses that are amplified during the initial infection.19 In addition, the majority of microbicide trials are being planned to take place in sub-Saharan Africa where the majority of infections occur through CCR5 viruses with a high prevalence of clade C viruses.20
MIV-150 is a nonnucleoside reverse transcriptase inhibitor (NNRTI) developed by Medivir AB (Huddinge, Sweden) for use as an antiviral therapeutic. The mechanism of action of NNRTIs is as an allosteric inhibitor of HIV reverse transcriptase (RT), which blocks viral DNA elongation thereby blocking viral replication. In vitro studies demonstrate 3 important characteristics of MIV-150. First, MIV-150 is a tight-binding HIV-RT enzyme inhibitor characterized by a rapid formation and slow dissociation rate and is effective at inactivating clinical isolates of HIV at very low concentrations. Second, Medivir scientists have demonstrated that MIV-150 inactivates viruses that are resistant to other antiviral drugs, including NNRTIs, nucleoside reverse transcriptase inhibitors (NRTIs), and protease inhibitors. Finally, resistance to MIV-150 develops much more slowly than resistance to several other currently marketed NNRTIs (Disa Bottinger, Bo Oberg, personal communication on MIV-150).
Because NNRTIs inhibit HIV strains independent of the virus phenotype, MIV-150 is likely to inhibit both T-tropic and M-tropic viruses. In support of this, studies were performed using clinical isolates from patients who have never been treated with NNRTIs and that represent HIV-1 clades A, B, C, and E. The activity of MIV-150 was similar against these clinical isolates from the 4 different subtypes of HIV. Additional studies carried out in the rhesus macaque model indicates that systemic injection of MIV-150 can prevent the onset of viremia after vaginal challenge with an M-tropic RT-SHIV (Disa Bottinger, Bo Oberg, personal communication).
Extensive toxicology studies carried out in mice, rats, dogs, and monkeys indicated that MIV-150 had no identified toxicity in the dose range studied. Human studies indicated that, although MIV-150 is well tolerated, it had poor systemic absorption after oral administration and, thus, is not suitable for antiviral therapy. Although the property of poor systemic absorption is a disadvantage for an oral therapeutic, it is an advantage for a microbicide that needs to be present and active in the vagina. Subsequently, under a contractual agreement, the Population Council was given exclusive rights for use of MIV-150 in a microbicide.
A series of experiments were conducted to confirm the active concentration of MIV-150 with those values established by Medivir to evaluate if MIV-150 can inactivate free virus and to determine if the combination of MIV-150 and Carraguard (PC-815) is more effective against both M-tropic and T-tropic HIV-1 than Carraguard. Further experiments compared Carraguard and PC-815 for activity against HIV-2 and activity in the presence of seminal fluid.
Materials and Methods
Preparation of PC-815
A 100 μmol/L stock solution of MIV-150 (Chiron Corporation, Emeryville, CA) was prepared using 100% ethanol. PC-815, which contains 10 μmol/L of MIV-150 and 3% carrageenan (PDR98-15; FMC BioPolymer, Philadelphia PA) in phosphate buffer was prepared by mixing desired concentrations of MIV-150 stock solution and carrageenan solution. Methylcellulose 2.5% in phosphate buffer was used as a negative control for antiviral activity testing.
Antiviral Activity of MIV-150
To evaluate the antiviral activity of MIV-150, the microtiter syncytial assay as described by Nara21 was used to determine the ability of MIV-150 to: 1) protect targets cell from becoming infected and 2) inactivate free virus. The assay uses CEM-SS cells, which were obtained from NIH AIDS Research and Reference Reagent Program (Rockville, MD). Cells were maintained in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS), 2 μmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco-BRL, Bethesda, MD).
Protection of Target Cells From Infection.
The HIV-1MN (NIH AIDS Research and Reference Reagent Program) virus inoculum (between 100 and 200 syncytial forming units [SFU]) and different concentrations of MIV-150 were simultaneously added to CEM-SS cells. After 3 to 4 days of infection, the number of SFU was quantified.
Inactivation of Free Virus.
Two hundred syncytial forming units of HIV-1MN were incubated with various concentrations of MIV-150 in a final volume of 200 μL of RPMI culture medium in a 96-well flat-bottomed plate (Falcon; BD Biosciences, San Jose, CA) for 1 hour at 37°C, 5% CO2, and 98% humidity. Centriprep YM-30 filtration columns (Millipore, Billerica, MA) were used to remove unbound MIV-150 from the viral inoculum as described by Motakis and Parniak.22 Briefly, 200 μL of viral inoculum and MIV-150 were diluted in 15 mL RPMI and centrifuged for 20 minutes at 1500 g at 4°C. The filtration step was repeated, and the viral inoculum was concentrated to approximately 500 μL. A volume of 150 μL of the viral inoculum was added to 50 μL of CEM-SS cells to perform the microtiter syncytial assay (4 × 104 cells/well).
The colorimetric assay based on the reduction of a tetrazolium salt (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide [XTT])23 was used to determine the cellular cytotoxicity of Carraguard and PC-815.
Additive Effect of MIV-150
Various concentrations of MIV-150 were mixed with different concentrations of carrageenan. The assay was used to identify any additive effect in the anti-HIV activity when MIV-150 is combined with carrageenan. The ability of the compounds to block HIV-1MN infection of TZM-bl cells (NIH AIDS Research and Reference Reagent Program) was obtained using the detection of β-galactoside activity in infected cells as described by Derdeyn et al.24
Activity Against Clinical Isolates of HIV
Primary clinical isolates (98/CN/009, 93/MW/959, 93/MW/960, 98/TZ/013, 98/TZ/017, 97/ZA/003, 97/ZA/009, 97/ZA/012, 93/MW/965, and 98/CN/006) of clade C HIV-1 (all of which are R5 viruses; however, 93/MW/959 and 93/MW/960 do not induce syncytia) were obtained from the NIH AIDS Research and Reference Reagent Program and were titered and evaluated in PBMC infection assay by monitoring the RT activity in the supernatant of infected cells as previously described.25 Carraguard and PC-815 were evaluated in this assay.
Strain HIV-2CDC310342 (NIH AIDS Research and Reference Reagent Program) is a primary isolate from an asymptomatic blood donor from the Cote D’Ivoire. The virus stock was 10−4/0.025 mL with an RT concentration of 10 ng/mL as determined by Lenti RT Activity Assay (Cavidi Tech, Uppsala, Sweden). Microbicidal activity of the different compounds was tested by using 100 TCID50 of virus in the presence of different concentrations of the compounds and activated PBMCs. The plate was then centrifuged at 2910 rpm for 99 minutes at 23°C and the wells washed 5 times with RPMI. Growth medium with 20 U/mL IL-2 was added next (NIH AIDS Research and Reference Reagent Program), and the plate was incubated at 37°C in 5% CO2 and 98% humidity. After 3 days, the RT activity in the supernatant was measured using a Lenti RT Activity Assay (Cavidi Tech) and the percentage about the virus control calculated. The experiment was repeated 3 times. The EC50 was calculated, and the median and standard deviation for the 3 experiments for MIV-150, Carraguard, and PC-815 was obtained.
Effect of Seminal Fluid on Anti-HIV Activity
Seminal fluid (SF) was obtained by centrifugation of semen, from healthy donors, to remove spermatozoa.26 Aliquots of SF were stored at −20°C until needed. To evaluate the full impact that SF may have on the activity of Carraguard and MIV-150, separate mixtures of undiluted SF were mixed with Carraguard, MIV-150, or phosphate-buffered saline (PBS; control). Additional controls were Carraguard or MIV-150 in PBS. Carraguard and MIV-150 were each diluted in SF or PBS at a concentration of 4 mg/mL and 400 μmol/L, respectively. They were then incubated for 30 minutes at 37°C. After incubation, mixtures were diluted to concentrations such that MIV-150 and Carraguard were still effective against HIV, but the toxicity and anti-HIV activity of SF were no longer present. The CEM-SS microtiter syncytial assay was performed to determine the effects of SF on the anti-HIV activity of MIV-150 and Carraguard.
In all experiments, each concentration of Carraguard, MIV-150, and PC-815 was tested in triplicate. The experiments were repeated at least twice. Standard deviations were calculated and plotted using Microsoft Excel software. Analysis of combined effects was done using the median effect principle. The combination index (CI) was calculated according to the method described by Chou and Talalay.27 The CI values are estimated using the Calcusyn for windows software package (Biosoft, Cambridge, U.K.): additive effect (CI = 1), synergism (CI <1), or antagonism (CI >1) for a given end point of the effect measurement.
Activity of MIV-150 Against HIV-1MN
To demonstrate that our laboratory obtained consistent results for the active concentration with those obtained by Medivir, the activity of MIV-150 to protect target cells from infection was assayed using a syncytial assay. The EC50 of MIV-150 against HIV-1MN in CEM.SS cells was below 0.001 μmol/L (Fig. 1). These data are in agreement with the findings of Medivir (Disa Bottinger, personal communication).
Activity of MIV-150 Against Free Virus
NNRTIs generally act after virus enters susceptible target cells where the RT is released in the cell as opposed to free virus where the RT enzyme is protected by the p24 capsid. To evaluate if MIV-150 inactivated free virus, the syncytial assay was used. MIV-150 effectively inactivated free virus with an EC50 of 0.004 μg/mL (or 0.01 μmol/L) (Fig. 2).
Additive Effect of PC-815
To determine whether there was an additive effect when MIV-150 was combined with Carraguard, various concentrations of each component were tested by detecting the β-galactoside activity in infected cells. The analysis of combined effects using the median effect principle showed values of CI around 1, supporting the idea of an additive effect (Fig. 3; Table 1).
Activity Against Clinical Isolates of HIV
The antiviral activity of Carraguard, PC-815, and methyl cellulose against clade C clinical isolates were compared. At the maximum concentration assayed, 160 μg/mL, methylcellulose had no effect. Carraguard had an EC50 of 77 μg/ml (range, 29–153 μg/mL) for 9 of the 10 clade C viruses tested. There was no effect on the 93/MW/959 virus strain at concentrations below 160 μg/mL. PC-815 blocked all 10 viruses with an EC50 of 7 μg/mL (range, 0.1–14 μg/mL). The average EC50 for PC-815 was approximately 10 times stronger than Carraguard (Table 2).
Activity Against HIV-2
Because HIV-2 infections are prevalent in some regions of West Africa, Carraguard, MIV-150, and PC-815 were assayed for antiviral activity against HIV-2 CDC310342 by quantifying the RT activity in the supernatant of infected cells. The EC50 of Carraguard was 10.5 μg/mL, EC50 of MIV-150 was 0.0003 μg/mL (or 0.001 μmol/L), and EC50 of PC-815 was 1.5 μg/mL (Fig. 4).
Effect of Seminal Fluid on Anti-HIV Activity
Because sexual transmission occurs in the presence of semen, MIV-150 and Carraguard were assayed in the presence of human SF. MIV-150 and Carraguard were assayed separately; the concentration of MIV-150 in PC-815 is very low and would be negligible when the formulation is diluted to offset the toxic effects of SF to cells in the syncytial assay. Toxicity and anti-HIV activity of SF had been determined in a previous experiment and found to be present when the culture medium contains more than 15% or 2% of SF, respectively (data not shown). SF had no effect on the antiviral activity of MIV-150 or Carraguard (Fig. 5A, B).
Although the mechanism(s) of the sexual transmission of HIV have yet to be fully understood, several theories suggest HIV can infect by either cell-associated or free virus.28–31 Given the evidence supporting several mechanisms of transmission, a microbicide capable of blocking infection by targeting different mechanisms of action will have a greater chance of being efficacious against viruses from a broader range of clades. It is also conceivable that a combination formulation may be active against other sexually transmitted pathogens.
MIV-150 was an attractive choice because of the extensive and encouraging data previously obtained with the compound. Scientists have demonstrated MIV-150 is highly effective against HIV and that the drug blocks viruses that are resistant to other marketed HIV antiretrovirals such as nevirapine, delavirdine, and efavirenz. In addition, resistance to MIV-150 develops more slowly than to other anti-HIV drugs tested (Medivir, Disa Bottinger, personal communication). For example, in vitro studies demonstrate that resistance development in the presence of nevirapine and delavirdine takes only 4 to 5 weeks compared with 45 weeks in the presence of MIV-150.
Additionally, in a recently held technical consultation jointly sponsored by the World Health Organization, International Partnership for Microbicides, and the Center for Disease Control and Prevention, it was recommended that the criteria for choosing candidate antiretrovirals for use in a microbicide include little to no cellular toxicity, low oral bioavailability, tight binding, high threshold to resistance development, activity against RT-resistant strains, antiviral activity at low concentrations, stable pH when exposed to a range of temperatures, and a short half-life. These properties are all characteristics of MIV-150.
Extensive animal and human toxicology studies have been carried out, all of which demonstrated that MIV-150 is safe, stable, and well tolerated. Also, it was demonstrated that the drug shows poor systemic absorption, an ideal characteristic for use in a microbicide. MIV-150 is inexpensive to manufacture, further increasing its appeal as a microbicide ingredient.
Data presented here show that Carraguard is effective against 9 of 10 clade C clinical isolates tested at a concentration of less than 160 μg/mL. However, when combined with MIV-150, PC-815 is effective against all clade C clinical isolates and is effective at concentrations an order of magnitude less than Carraguard alone. The actual concentration of carrageenan in undiluted Carraguard is 30,000 μg/mL (approximately 200 times more than the highest concentration used in the PBMC assay). Thus, Carraguard alone displayed strong antiviral activity. Even so, based on these findings, PC-815 should be more effective than Carraguard in preventing infection by clade C viruses.
Microbicide efficacy against HIV-2 is important because although infections by this virus are mainly restricted to West Africa, the prevalence is growing in some areas of Africa, Europe, and Southwest India.32 Some NNRTIs that are effective against HIV-1 are not effective against HIV-2.33 HIV-1 and HIV-2 differ in virus evolution, tropism, and pathogenesis. HIV-2 demonstrates lower pathogenicity compared with HIV-1.32 Related to this lower pathogenicity is an enhanced immune control of HIV-2 infection. Furthermore, CD4-independent infection of target cells can occur with HIV-2, whereas CD4 receptors are integral to infection of main targets (lymphocyte T CD4+ cells and macrophages) for HIV-1.32
All of the formulations tested demonstrated in vitro antiviral activity against HIV-2CDC310342. The in vitro activity of PC-815 against HIV-2 suggests the possibility that the formulation could be evaluated in vivo for activity against SIV or SHIV using the macaque model because HIV-2 is more similar to SIV/SHIV phylogenetically than HIV-1.34 This would be a valuable avenue for future research.
Theoretically, MIV-150 could prevent sexual transmission of HIV in 3 ways. The first would be to enter target cells in the submucosa, bind to and inactive the RT enzyme after the viral capsid has been uncoated. In free virus, the enzyme is associated with the viral genome and is protected by the viral capsid and envelope. However, using the CEM-syncytial assay MIV-150 inactivated free HIV-1MN. This suggests a second and possibly third way by which MIV-150 could prevent HIV infection by either entering a virion and inactivating the RT or by associating with the virion and being carried into a target cell with the virion and subsequently inactivating the RT. Thereby, it could be effective against virus in the vagina before viral infection of the target cells. This is especially important in the case of sexual transmission of HIV because considerable evidence suggests that free virus is captured by dendritic cells and transferred to T-cells locally and/or transported to the lymph nodes.35
Sexual transmission occurs in the presence of SF. It is, therefore, important to determine whether SF will inactivate the active ingredients in the microbicide. In testing possible interference of SF by testing anti-HIV activity of MIV-150 or carrageenan in the presence or absence (control) of human SF, we found that SF had no effect.
In summary, the effectiveness of Carraguard has been improved by adding the NNRTI, MIV-150. The combination product has greater strength against clade C clinical isolates and HIV-2, acts on free virus, and is not inactivated by SF.
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